Vitamin D is a fat-soluble secosteroid that is responsible for intestinal absorption of calcium and phosphate. The metabolic pathway for Vitamin D is complex and involves multiple mono-, di- and tri-hydroxy-forms of both Vitamin D3 and Vitamin D2 together with a number of epimer and “pre” isomers. In humans, Vitamin D3 is synthesized in the skin by the action of sun-light while a relatively small amount of vitamin D2 is obtained from dietary sources. In some cases, pharmaceutical Vitamin D2 might be taken as a supplement. In the liver, Vitamin D is converted to 25-hydroxyvitamin D (abbreviated 25-OH-Vit-D) and current clinical practice uses the total serum 25-OH-Vit-D (i.e., 25-OH-Vit-D3 plus 25-OH-Vit-D2) levels to assess a person's Vitamin D status. The 25-OH-Vit-D metabolite is then converted primarily in the kidneys to the hormonally active form 1,25-dihydroxyvitamin D (abbreviated 1,25-(OH)2-Vit-D). 1,25-(OH)2-Vit-D circulates as a hormone in the blood and is responsible for regulating the concentration of calcium and phosphate in the bloodstream to promote healthy growth and remodeling of bone. 25-OH-Vit-D is also converted to 1,25-(OH)2-Vit-D outside of the kidneys for other purposes, such as in the proliferation, differentiation, and apoptosis of cells.
Other metabolites also prove to be informative and relevant to the overall biological effect of Vitamin D. For example, while the metabolite 24,25-(OH)2-Vit-D alone has no known biological activity, it represents the first step in the pathway to degrade 25-OH-Vit-D and is not routinely measured. The enzyme responsible (Vitamin D 24-hydroxylase; CYP24A1) plays an important role in maintaining a normal (“sufficient”) concentration of 25-OH-Vit-D. Too high a concentration of 25-OH-Vit-D can cause hypercalcemia and recent evidence suggests that at least some cases of Idiopathic Infantile Hypercalcemia (IIH) might be the result of natural polymorphisms in the CYP24A1 gene.
It has been recently recognized that a structural isomer of 25-OH-Vit-D3 (the C3-epimer), which was thought only to be present in pediatric samples, is also present in adults. C3-epi-25-OH-Vit-D3 has no known biological relevance and it should therefore not be included in the measurement of Vitamin D status. Recent studies have highlighted that the majority of LC/MS/MS assays used by laboratories to report results to the Vitamin D External Quality Assurance Scheme (DEQAS) are not designed to differentiate between 25-OH-Vit-D3 and the C3-epimer. This is because the two isomers (25-OH-Vit-D3 and the C3-epimer) have identical chemical composition, molecular mass and MS/MS characteristics such that they cannot be separated solely on the basis of mass spectrometry. Instead, both isomers must first be separated from each other before they are introduced into the analyser region of the mass spectrometer. This can be achieved using conventional liquid chromatography. However, this difficult separation can take, if at all, from approximately 6 minutes to approximately 12 minutes per sample, which makes routine adoption of LC/MS based assays in a clinical laboratory impractical. Although some immunoassays are able to differentiate between 25-OH-Vit-D3 and the C3-epimer, there are other interferences and difficulties which result in most immunoassays having relatively poor assay characteristics (accuracy, precision, linearity etc.). Thus, at present there appears to be no single assay for Vitamin D status that provides all the desirable assay characteristics.
LC/MS analyses can quantitatively measure multiple analytes in the same analytical run. For example, in addition to analyzing 25-OH-Vit-D2 and 25-OH-Vit-D3 separately from C3-epi-25-OH-Vit-D3, an LC/MS method can also measure other Vitamin D metabolites (e.g., 24,25-(OH)2-Vit-D, 1,25-(OH)2-Vit-D, etc.) in the same analysis. Such a multi-analyte analysis can provide a much more robust Vitamin D status. However, the analysis times of an LC/MS Vitamin D metabolite panel such as this becomes lengthy and impractical for routine laboratories.
In addition, because some Vitamin D metabolites are present at low concentrations in serum and plasma (e.g., 1,25-(OH)2-Vit-D, 1,24,25-(OH)3-Vit-D, etc), derivatization methods with Cookson-type reagents (e.g., 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and DMEQ-TAD (4-[2-(3,4-Dihydro-6,7-dimethoxy-4-methyl-3-oxo-2-quinoxalinyl)ethyl]-3H-1,2,4-triazole-3,5(4H)-dione)) to form, e.g., PTAD-linked Vitamin D metabolites derivatives, have been used as a way to increase the sensitivity of detecting these metabolites by mass spectrometry. However, this derivatization process results in pairs of isomeric derivatives for each analyte and may create inferences. Therefore, a significant challenge has been to resolve, in a short analysis time compatible with high sample throughput, these low concentration derivatives under current chromatographic techniques.